Methods of treating diabetes and/or promoting survival of pancreatic islets after transplantation

ABSTRACT

Disclosed herein are methods for treating/and or preventing diabetes using a specific inhibitor of SMAD7 expression or function. Also disclosed are methods of promoting organ and/or cell, e.g., pancreatic islet cell, survival after transplantation using a specific inhibitor of SMAD7 expression or function.

CROSS REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage of International (PCT)Patent Application No. PCT/US2013/037150, filed Apr. 18, 2013, whichclaims priority to and the benefit of U.S. provisional application61/625,904, filed Apr. 18, 2012, the entire contents of which are hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention is generally directed to antisenseoligonucleotides against SMAD7 and uses thereof in treating and/orpreventing diabetes and/or in promoting pancreatic islet cell survivalafter transplantation.

BACKGROUND

Diabetes is a metabolic disease characterized by high levels of sugar inthe blood. The two most prevalent types of diabetes are Type 1 diabetesmellitus (T1DM) and Type 2 diabetes mellitus (T2DM). Type 1, orinsulin-dependent diabetes mellitus (IDDM), is a chronic autoimmunedisease characterized by the extensive loss of beta cells in thepancreatic islets, which produce insulin. Type 2 diabetes, ornon-insulin dependent diabetes mellitus (NIDDM), develops when muscle,fat and liver cells fail to respond normally to insulin (insulinresistance). In particular, Type 2 diabetes has become an epidemic,driven by increases in obesity and a sedentary lifestyle, and thegeneral aging of the populations in many countries. In recent clinicalpractice, it has become increasingly difficult to distinguish T1DM fromT2DM as many children with T1DM are overweight at diagnosis, and aconsiderable proportion of physician-diagnosed T2DM youth have evidenceof pancreatic autoimmunity (Badaru, A. and Pihoker, C. “Type 2 diabetesin childhood: clinical characteristics and role of beta-cellautoimmunity,” Curr. Diab. Rep., 2012, 12, 75-81). In 2011, more than346 million people worldwide were affected by diabetes.

Recent studies have demonstrated that TGF-β plays a role in pancreaticislet function and diabetes development (Moritani, M. et al. “Abrogationof autoimmune diabetes in nonobese diabetic mice and protection againsteffector lymphocytes by transgenic paracrine TGF-β1,” J. Clin. Invest.,1998, 102, 499-506; Olivieri, A. et al. “Serum transforming growthfactor β1 during diabetes development in non-obese diabetic mice andhumans,” Clin. Exp. Immunol., 2010, 162, 407-414). For example, anislet-specific pulse of TGF-β expression for one week has been shown todelay diabetes development in NOD mice (Wållberg, M. et al. “Anislet-specific pulse of TGF-β abrogates CTL function and promotes β cellsurvival independent of Foxp3⁺ T cells,” J. Immunol., 2011, 186,2543-2551), a commonly used animal model of type 1 diabetes (Roep, B. O.et al. “Satisfaction (not) guaranteed: re-evaluating the use of animalmodels of type 1 diabetes,” Nat. Rev. Immunol., 2004, 4, 989-997).TGF-β1 was effective not only in curing diabetes in diabetic NOD miceand blocking islet destructive autoimmunity, but also in inducing isletregeneration (Luo, X. et al. “Systemic transforming growth factor-β genetherapy induces Foxp3+ regulatory cells, restores self-tolerance, andfacilitates regeneration of beta cell function in overtly diabeticnonobese diabetic mice,” Transplantation, 2005, 79, 1091-1096). Hence,therapeutic interventions along this pathway, may not only stop theprogression of the disease, but might even restore function (i.e.,adequate insulin production) after onset of hyperglycemia.

The TGF-βs 1-3 are involved in a variety of biological functionsincluding cell growth, organ development, fibrogenesis, and regulationof immune cells. TGF-β1 is the predominant form expressed in the immunesystem, and it is now well-recognized as a critical regulator in immuneresponses that can dampen T cell responses (Li, M. O. and Flavell, R. A.“TGF-beta: a master of all T cell trades,” Cell, 2008, 134, 392-404).Specifically, TGF-β1 binds a heterodimeric transmembraneserine/threonine kinase receptor containing two subunits, TGF-β1 R1 andTGF-β1 R2. Upon ligand binding, the TGF-β1 R1 receptor is phosphorylatedby the constitutively active TGF-β1 R2 receptor and signal is propagatedto the nucleus by proteins belonging to the SMAD family. ActivatedTGF-β1 R1 directly phosphorylates SMAD2 and SMAD3 proteins, which theninteract with SMAD4. The complex of SMAD2/SMAD3/SMAD4 translocates tothe nucleus and modulates the transcription of certain genes. SMAD7 isanother member of this protein family that acts as a general antagonistfor TGF-β through negative-feedback mechanisms (Yan, X. and Chen, Y. G.“Smad7: not only a regulator, but also a cross-talk mediator of TGF-betasignalling,” Biochem. J., 2011, 434, 1-10).

Studies have demonstrated that SMAD7 plays a role in diabetes and β-cellfunction. SMAD7, an intracellular protein, has been shown to interferewith binding of SMAD2/SMAD3 to the TGF-β1 R1 preventing phosphorylationand activation of these proteins, leading to inhibition of TGF-β1mediated-signaling. Expression of SMAD7 in pancreatic β-cells has beenshown to disrupt TGF-β signaling and induce reversible diabetes mellitus(Smart, N. G. et al. “Conditional expression of Smad7 in pancreatic betacells disrupts TGF-beta signaling and induces reversible diabetesmellitus,” PLoS Biol., 2006, 4, e39). Furthermore, results in NOD micealso implicate the Smad2 and TGF-β signaling pathway in activateddendritic cells in diabetogenesis, and there is evidence from humangenome-wide association studies supporting a role for Smad7 in humantype 1 diabetes (Rook, S. M. et al. “Smad2: A candidate gene for themurine autoimmune diabetes locus Idd21.1,” J. Clin. Endocrinol. Metab.,2011, 96, E2072-E2077). Since TGF-β1 has been shown to contribute to thesuppression of cytokine production, the inhibition of T cell response,and the induction of regulatory T cells (Treg) (Kawamoto, K. et al.“Transforming growth factor beta 1 (TGF-β1) and rapamycin synergize toeffectively suppress human T cell responses via upregulation of FoxP3⁺Tregs,” Transpl. Immunol., 2010, 23, 28-33), SMAD7 modulation could alsobe beneficial in islet transplantation by supporting graft function,limiting toxicity, and preventing immune rejection.

Thus, there is an unmet need for new therapies in diabetes andpancreatic islet transplantation.

SUMMARY

The disclosed methods are based, in part, upon the discovery of specificinhibitors of SMAD7 expression or function, e.g., antisenseoligonucleotides against SMAD7, that inhibit SMAD7 and therefore restoreTGF-β signaling. Exemplary antisense oligonucleotides against SMAD7include, for example, SEQ ID No:5 or SEQ ID No: 6, which may be used ina pharmaceutical composition. The disclosed SMAD7 inhibitors can be usedto treat and/or prevent diabetes, e.g., Type 1 diabetes, Type 2diabetes, and gestational diabetes. The antisense oligonucleotidesagainst SMAD7 can also be used to promote pancreatic islet cell survivalafter transplantation.

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following figures, detailed descriptionand claims. As used herein, “including” means without limitation, andexamples cited are non-limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 (A) provides the nucleic acid sequence of SMAD7 (SEQ ID NO: 1)and (B) provides the amino acid sequence of SMAD7 (SEQ ID NO: 2).

FIG. 2 (A) is a graph showing the effects of TGFβ and GED-0301 (a SMAD7antisense oligonucleotide, SEQ ID NO: 6) on NF-κB activation in HEK-BlueTNF-α sensor cells and (B) is a graph showing the effect of TGFβ andGED-0301 on NF-κB activation in HEK-Blue CD40L sensor cells challengedby TNF-α and CD40L, respectively.

FIG. 3 (A) is a panel illustrating the gating methodology used inanalysis of flow cytometry results for the organ-specific uptake offluorescein-labeled GED-0301 following administration by various routesin mice and FIG. 3 (B) shows corresponding percent uptakes (FITC+) inviable cells in the indicated organ tissues.

FIG. 4 is a multichannel high resolution microscopic image depictingsections of pancreas from a control mouse and a mouse administeredfluorescein-labeled GED-0301 subcutaneously (4 hours afteradministration). Color legend: green—fluorescent GED-0301, grey—DAPI (amarker of cell nucleus), red—insulin (indicating insulin-producing(β-cells), and blue—CD45 (indicating CD45 or leukocyte common antigen,LCA, a marker of white blood cells, the elements of the circulatingblood system that comprise the cells of immunity and inflammation).

FIG. 5(A) is a graph depicting blood glucose levels in NOD mice treatedwith the SMAD7 antisense oligonucleotide GED-0301 (AS GED-0301), thecorresponding sense control (S control), and saline (Control) (125μg/animal, subcutaneous (s.c.), daily) following onset of hyperglycemiaand FIG. 5 (B) is a corresponding Kaplan-Meier survival curve showingthe percent of animals remaining diabetes-free as a function of time.

FIG. 6 is a graph comparing the results of an oral glucose tolerancetest conducted in GED-0301-treated NOD mice that did not become diabeticby the end of the study (day 150) and control mice.

FIG. 7 shows multichannel high resolution Z-stacks of confocalmicroscopic images comparing representative islet-containing sections ofpancreas from GED-0301-treated NOD mice that did not become diabetic bythe end of the study (day 150) to those of corresponding controls. Colorlegend: blue—DAPI (a marker of cell nucleus), red—insulin (indicatinginsulin-producing β-cells), and green—glucagon (indicatingglucagon-producing α-cells).

DETAILED DESCRIPTION

The present disclosure is generally directed to antisenseoligonucleotides against SMAD7 and uses thereof in treating and/orpreventing diabetes and/or in promoting pancreatic islet cell survivalafter transplantation.

Anti-SMAD7 Therapy

The disclosed methods relate to the use of a specific SMAD7 inhibitorselected from the group consisting of small binding molecules, e.g.,natural and synthetic compounds, antibodies, aptamers, intramers, RNAi(double stranded RNA, siRNA) and anti-SMAD7 antisense molecules fortreating and/or preventing diabetes and/or in promoting pancreatic isletcell survival after transplantation. SMAD7 inhibitors may also comprisetruncated and/or mutated SMAD7 molecules which interfere with the SMAD7and which, thereby, inhibit SMAD7 function.

For example, anti-SMAD7 therapy includes targeted therapies againstSMAD7 (e.g., anti-SMAD7 antisense therapies, i.e., antisenseoligonucleotide against SMAD7, and antibodies against SMAD7). Antisenseoligonucleotides are short synthetic oligonucleotide sequencescomplementary to the messenger RNA (mRNA), which encodes for the targetprotein (e.g., SMAD7). Antisense oligonucleotide sequences hybridize tothe mRNA producing a double-strand hybrid that can lead to theactivation of ubiquitary catalytic enzymes, such as RNase H, whichdegrades DNA/RNA hybrid strands thus preventing protein translation.

In certain embodiments, an anti-SMAD7 antisense oligonucleotide maytarget site 403, 233, 294, 295, 296, 298, 299, and/or 533 (i.e.,nucleotides 403, 233, 294, 295, 296, 298, 299, and 533, respectively) ofthe human SMAD7 mRNA (e.g., of SEQ ID NO: 1) (see FIG. 1). In anexemplary embodiment, the anti-SMAD7 antisense oligonucleotide targetsnucleic acids 403-423 of human SMAD7 mRNA.

In certain embodiments, an antisense oligonucleotide may be derived fromthe following anti-SMAD7 antisense oligonucleotide5′-GTCGCCCCTTCTCCCCGCAGC-3′ (SEQ ID NO: 3).

It is contemplated herein that an antisense oligonucleotide targetingSMAD7 may comprise a mixed-backbone wherein the cytosine residues in aCpG pair are replaced by 5′-methylcytosine (abbreviated as Me-dC).Methylphosphonate linkages may also be placed at the 5′ and/or 3′ endsof an antisense oligonucleotide (abbreviated as MeP). The phosphonatebackbone of a contemplated anti-SMAD7 antisense oligonucleotide mayoptionally include 1, 2, 3, 4 or more phosphorothioate bonds (e.g.,phosphorothioate bonds would replace phosphodiester bonds). In anembodiment, all phosphonate bonds may be phosphorothioate bonds.

Exemplary antisense oligonucleotide therapies that target SMAD7 include,but are not limited to:

5′-GTXYCCCCTTCTCCCXYCAG-3′ (SEQ ID NO: 4), wherein X is a nucleotidecomprising a nitrogenous base selected from the group consisting ofcytosine and 5-methylcytosine or a 2′-O-methylcytosine nucleoside, andwherein Y is a nucleotide comprising a nitrogenous base selected fromthe group consisting of guanine and 5-methylguanine or a2′-O-methylguanine nucleoside, provided that at least one of thenucleotides X or Y comprises a methylated nitrogenous base;

5′-GTXGCCCCTTCTCCCXGCAG-3′ (SEQ ID NO: 5), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate;

5′-GTXGCCCCTTCTCCCXGCAGC-3′ (SEQ ID NO: 6), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate;

5′-ZTXGCCCCTTCTCCCXGCAZ-3′ (SEQ ID NO: 7), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylphosphonate;

5′-ZTXGCCCCTTCTCCCXGCAZC-3′ (SEQ ID NO: 8), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylphosphonate.

In a particular embodiment, contemplated SMAD7 antisense may be asequence comprising one of:

5′-GTXGCCCCTTCTCCCXGCAG-3′ (SEQ ID NO: 9), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphorothioate;

5′-GTXGCCCCTTCTCCCXGCAGC-3′ (SEQ ID NO: 10), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphorothioate;

5′-ZTXGCCCCTTCTCCCXGCAZ-3′ (SEQ ID NO: 11), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylthiophosphonate;

5′-ZTXGCCCCTTCTCCCXGCAZC-3′ (SEQ ID NO: 12), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylthiophosphonate.

For example, SEQ ID NOs. 9-12 include 1, 2, 3, 4 or morephosphorothioate bonds. In an embodiment, all O,Ophosphonate bonds ofSEQ ID NOs. 9-12 are phosphorothioate bonds.

Methods of Treatment

Provided herein are methods of promoting organ and/or cell, for example,pancreatic islet cell, survival after transplantation, comprisingadministering to a patient in need thereof an effective amount of aspecific inhibitor of SMAD7 expression or function, for example, anantisense oligonucleotide against SMAD7.

Also, provided herein are methods of treating and/or preventingdiabetes, e.g., Type 1 diabetes and Type 2 diabetes, comprisingadministering to a patient in need thereof an effective amount of aspecific inhibitor of SMAD7 expression or function, for example, anantisense oligonucleotide against SMAD7.

Diabetes includes a group of metabolic diseases characterized by highblood sugar or ketoacidosis, as well as chronic, general metabolicabnormalities arising from a prolonged high blood sugar status or adecrease in glucose tolerance. Diabetes includes both the Type 1 andType 2 forms of the disease, gestational diabetes, and other-conditionsof insulin deficiency.

It will be appreciated that the disclosed methods are also applicable totreating and preventing latent autoimmune diabetes (i.e., type 1.5diabetes), metabolic imbalances, a pre-diabetic state, metabolicsyndrome, a lipid or glucose related disorder, e.g., hyperlipidemiaand/or hypercholesterolemia, and other related disorders.

Metabolic imbalances can include any disorder or disease state orcondition that are associated with an elevated plasma glucose. Ametabolic imbalance, for example, comprises diabetes mellitus,gestational diabetes, genetic defects of β-cell function, geneticdefects in insulin action, diseases of the exocrine pancreas,endocrinopathies, drug or chemical-induced infections, other geneticsyndromes associated with diabetes, a pre-diabetic state, and metabolicsyndrome.

Metabolic syndrome is characterized by a group of metabolic risk factorsin one person, as described by the American Heart Association (AHA).Metabolic syndrome is also known as metabolic syndrome X, syndrome X,insulin resistance syndrome, Reaven's syndrome, or CHAOS. The riskfactors include, but are not limited to, abdominal obesity, atherogenicdyslipidemia, hypertension, insulin resistance or glucose intolerance,prothrombotic state (high fibrinogen or plasminogen activatorinhibitor-1), and proinflammatory state (elevated C-reactive protein).

The terms “treat”, “treatment”, “treating” and the like are used hereinto generally mean obtaining a desired pharmacological and/orphysiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of partially or completely curing a diseaseand/or adverse effect attributed to the disease. The term “treatment” asused herein covers any treatment of a disease in a mammal, particularlya human, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e. arresting itsdevelopment; or (c) relieving the disease, i.e. causing regression ofthe disease.

In certain embodiments, a subject's responsiveness to treatment with ananti-SMAD7 therapy can be interpreted with respect to a control sample(described below) obtained from the subject prior to treatment. Asubject may be identified as responding to treatment with an anti-SMAD7therapy if there is a reduction in SMAD7 expression; if there is anincrease in the total pancreatic insulin content; if β-cell function ismaintained and/or restored (for example, as assessed via standardmethods such as the intravenous glucose tolerance test, IVGTT); and/orif normoglycemic, i.e., normal glucose content of the blood, ismaintained and/or restored. In other embodiments, a subject which hasreceived an islet graft, e.g., an allogenic islet graft, may beidentified as responding to treatment with anti-SMAD7 therapy if thereis prevention of rejection and/or prolonged survival of the islets.

A test sample may be obtained from the patient, for example, at week 1,week 2, week 4, week 8, week 10 or later after initiation of therapy todetermine sensitivity to treatment. In some embodiments, the test samplemay be obtained, for example, one week, two weeks, three weeks, fourweeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, tenweeks, eleven weeks, twelve weeks, five months, six months, and/or oneyear or longer after the initiation of therapy to monitor sensitivity totreatment.

A control sample may include a sample (e.g., a blood or tissue sample)obtained from the subject prior to treatment with an anti-SMAD7 therapy.The control sample provides a baseline for monitoring a subject'sprogress to treatment. A control sample may be obtained from the subjecton the day the anti-SMAD7 therapy is first administered (e.g., Day 1 ofa treatment regimen). In other embodiments, a control sample may beobtained from a subject one day prior to the start of an anti-SMAD7therapy (e.g., Day 0 of a treatment regimen). Alternatively, a controlsample may be obtained from a subject 2, 3, 4, 5, 6, 7 or more daysprior to the start of an anti-SMAD7 therapy. For example, theupregulation or down regulation of certain cell samples may be measuredprior to treatment (e.g., a control sample), during treatment, and/orafter treatment to monitor a subject's response to therapy, e.g., ananti-SMAD7 therapy.

A control sample may include, for example, a sample to monitor theglucose level of a subject, wherein the sample was obtained from thesubject prior to treatment with an anti-SMAD7 therapy.

In some embodiments, a control level may be established for a subjectbased on long-term monitoring of certain cell populations in thesubject. In such instances, it is contemplated that a subject mayundergo multiple rounds of treatment with an anti-SMAD7 therapy. Theamount of a certain cell population detected following multiple roundsof treatment may be compared to a prior control level for the subject todetermine whether the subject has responded to therapy and/or is likelyto respond to further treatment with an anti-SMAD7 therapy. In otherembodiments, a control or baseline level for a subject may beestablished based on an average measurement of a certain cell populationdetermined from multiple baseline samples obtained over time (e.g.,obtained over the course of weeks, months, or years). Accordingly, anytest or assay conducted as disclosed herein may be compared with aprevious or established control level and it may not be necessary toobtain a new control sample from the subject for comparison, e.g., ifthe subject is receiving more than one round of treatment with ananti-SMAD7 therapy.

Administration and Formulation

In some embodiments, contemplated herein are pharmaceutical compositionscomprising a contemplated antisense oligonucleotide against SMAD7 and apharmaceutically acceptable carrier. For example, the pharmaceuticalcomposition may be administered subcutaneously. Alternatively, thepharmaceutical composition may be administered orally.

As used herein, “pharmaceutically acceptable carrier” means buffers,carriers, and excipients suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The carrier(s) should be “acceptable” inthe sense of being compatible with the other ingredients of theformulations and not deleterious to the recipient. Pharmaceuticallyacceptable carriers include buffers, solvents, dispersion media,coatings, isotonic and absorption delaying agents, and the like, thatare compatible with pharmaceutical administration. The use of such mediaand agents for pharmaceutically active substances is known in the art.

In one embodiment, a contemplated antisense oligonucleotide againstSMAD7 and any pharmaceutical composition thereof may be administered byone or several routes, including orally, topically, parenterally, e.g.,subcutaneous injection, by inhalation spray or rectally. The termparenteral as used herein includes subcutaneous injections,intrapancreatic administration, intravenous, intramuscular,intraperitoneal, intrasternal injection or infusion techniques. Forexample, the antisense oligonucleotide against SMAD7 may be administeredsubcutaneously to a subject. In another example, the antisenseoligonucleotide against SMAD7 may be administered orally to a subject.

Pharmaceutical compositions containing an antisense oligonucleotideagainst SMAD7, such as those disclosed herein, can be presented in adosage unit form and can be prepared by any suitable method. Apharmaceutical composition should be formulated to be compatible withits intended route of administration. Useful formulations can beprepared by methods well known in the pharmaceutical art. For example,see Remington's Pharmaceutical Sciences, 18th ed. (Mack PublishingCompany, 1990).

Pharmaceutical formulations preferably are sterile: Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

In an exemplary embodiment, a pharmaceutical composition forsubcutaneous administration of an antisense oligonucleotide againstSMAD7 comprises an antisense oligonucleotide such as that represented bySEQ ID NO: 6, or a pharmaceutically acceptable salt thereof (such as asodium salt), and a pharmaceutically acceptable carrier.

In another embodiment, a pharmaceutical tablet formulation for oraladministration of an antisense oligonucleotide against SMAD7 comprisesan intragranular phase, wherein the intra-granular phase includes anantisense oligonucleotide such as that represented by SEQ ID NO: 6, or apharmaceutically acceptable salt thereof (such as a sodium salt), and apharmaceutically acceptable filler, and which may also include anextra-granular phase, that may include a pharmaceutically acceptableexcipient such as a disintegrant. For example, a pharmaceuticallyacceptable tablet for oral use may comprise an intra-granular phase,comprising about 5 to about 10% by weight antisense oligonucleotiderepresented by SEQ ID NO 6 or a pharmaceutically acceptable saltthereof, about 40% by weight mannitol, about 8% by weightmicrocrystalline cellulose, about 5% by weight hydropropylmethylcellulose, and about 2% by weight sodium starch glycolate; anextra-granular phase comprising about 17% by weight microcrystallinecellulose, about 2% by weight sodium starch glycolate, about 0.4% byweight magnesium stearate; and an enteric coating over the tablet,comprising about 13% by weight AcyrlEZE® (see, e.g., PCT Publication No:WO/2010/054826, which is hereby incorporated by reference in itsentirety).

Exemplary formulations include dosage forms that include or consistessentially of about 35 mg to about 500 mg of an antisenseoligonucleotide against SMAD7. For example, formulations that includeabout 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg,120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg,or 250 mg of an antisense oligonucleotide against SMAD7 are contemplatedherein. In one embodiment, a formulation may include about 40 mg, 80 mg,or 160 mg of an antisense oligonucleotide against SMAD7. The amountadministered will depend on variables such as the type and extent ofdisease or indication to be treated, the overall health of the patient,the in vivo potency of the antibody, the pharmaceutical formulation, andthe route of administration. The initial dosage can be increased beyondthe upper level in order to rapidly achieve the desired blood-level ortissue level. Alternatively, the initial dosage can be smaller than theoptimum, and the dosage may be progressively increased during the courseof treatment. Human dosage can be optimized, e.g., in a conventionalPhase I dose escalation study designed to run from 40 mg to 160 mg.Dosing frequency can vary, depending on factors such as route ofadministration, dosage amount and the disease being treated. Exemplarydosing frequencies are once per day, once per week and once every twoweeks. In some embodiments, dosing is once per day for 7 days. Apreferred route of administration is subcutaneous.

In some embodiments, methods provided herein may further includeadministering at least one other agent that is directed to treatment ofdiseases and disorders disclosed herein. In one embodiment, contemplatedother agents may be co-administered (e.g., sequentially orsimultaneously).

Agents contemplated include immunosuppressive agents includingglucocorticoids, cytostatics, antibodies, agents acting onimmunophilins, interferons, opioids, TNF binding proteins,mycophenolate, and small biological agents. For example, contemplatedimmunosuppressive agents include, but are not limited to: tacrolimus,cyclosporine, pimecrolimus, sirolimus, everolimus, mycophenolic acid,fingolimod, dexamethasone, fludarabine, cyclophosphamide, methotrexate,azathioprine, leflunomide, teriflunomide, anakinra, anti-thymocyteglobulin, anti-lymphocyte globulin, muromonab-CD3, afutuzumab,rituximab, teplizumab, efalizumab, daclizumab, basiliximab, adalimumab,infliximab, and etanercept.

Agents contemplated include drug therapies for regulating blood sugarlevels including oral therapies with hypoglycemic agents and/or oralanti-diabetic agents, injectable therapies, and the like. Non-drugtherapies for regulating blood sugar level include, but are not limitedto, diatetic and/or exercise control measures.

Oral drug therapies for regulating blood sugar levels includehypoglycemic agents that may include, but are not limited to: Acarbose,Acetohexamide, Chlorpropamide, Darglitazone Sodium, Glimepiride,Glipizide, Glyburide, Repaglinide, Troglitazone, Tolazamide, andTolbutamide.

Oral drug therapies for regulating blood sugar levels includeantidiabetic agents that may include but are not limited to: Acarbose,Acetohexamide, Buformin, Butoxamine Hydrochloride, Camiglibose,Chlorpropamide, Ciglitazone, Englitazone Sodium, EtoforminHydrochloride, Gliamilide, Glibornuride, Glicetanile Gliclazide Sodium,Gliflumide, Glipizide, Glucagon, Glyburide, Glyhexamide, GlymidineSodium, Glyoctamide, Glyparamide, Insulin Isophane, Insulin Human Zinc,Extended Insulin, Insulin Lispro, Linogliride, Linogliride Fumarate,Metformin, Methyl Palmoxirate, Palmoxirate Sodium, PioglitazoneHydrochloride, Pirogliride Tartrate, Proinsulin Human, Repaglinide,Seglitide Acetate, Tolazamide, Tolbutamide, Tolpyrramide, Troglitazone,and Zopolrestat.

Injectable therapies for regulating blood sugar levels may include, butare not limited to fast-acting insulin, long-acting insulin, and relatedinsulin. Fast-acting insulin includes regular insulin, Prompt InsulinZinc Suspension, and Semilente® insulin, Humalog® Injection, Humulin® R,Iletin II, Novolin R, Purified Pork Regular Insulin, and Velosulin BRHuman Insulin. Long-acting insulin includes Protamine Zinc InsulinSuspension, Extended Insulin Zinc Suspension, Ultralente® Insulin, andHumulin® U. Other insulin includes Isophane Insulin Suspension, NPHinsulin, isophane insulin; Insulin Zinc Suspension Lente® Insulin, HumanInsulin Isophane Suspension/Human Insulin Injection, Humulin® 50/50,Humulin® 70/30, and Novolin® 70/30.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only, and are not to beconstrued as limiting the scope or content of the invention in any way.

Example 1 Effect of SMAD7 Antisense on NF-κB Activation

HEK-Blue™ (InvivoGen) TNF-α/IL-1β sensor cells are designed to detectbioactive TNF-α and IL-1β by monitoring the activation of the NF-κBpathway. These cells were generated by stable transfection of humanembryonic kidney HEK293 cells with a SEAP reporter gene under thecontrol of the IFN-β minimal promoter fused to five NF-κB and five AP-1binding sites. HEK-Blue™ (InvivoGen) CD40L sensor cells are used tomeasure the bioactivity of CD154 (CD40L) through the secretion ofembryonic alkaline phosphatase (SEAP) upon NF-κB activation followingCD40 stimulation. These cells were generated by stable transfection ofHEK293 cells with the human CD40 gene and an NF-κB-inducible SEAPconstruct. Therefore, HEK-Blue™ cells measure the bioactivity ofTNFα/CD40L through the secretion of embryonic alkaline phosphatase(SEAP) upon NF-κB activation following TNFR/CD40 stimulation. Tumornecrosis factor alpha (TNF-α) is a known cytokine involved in systemicinflammation and the regulation of immune cells. CD40L is aco-stimulatory protein involved in T-cell activation and the developmentof effective immune responses, and is thought to be involved in thedevelopment of autoimmune diseases including T1DM (Margolles-Clark, E.et al. “Small molecule costimulatory blockade: organic dye inhibitors ofthe CD40-CD154 interaction,” J. Mol. Med., 2009, 87, 1133-1143).

HEK Blue TNFα and CD40L sensor cells (Invivogen) (50,000/well) wereincubated with GED-0301 antisense (AS) or control sense (S)oligonucleotide (OGN; 4 μg/mL) for 6 h in the presence of lipofectamine(LPF) then media for 18 h followed by challenge by TNFα (1 ng/mL) orCD40L (25 ng/mL) in the presence or absence of TGF-β (200 ng/mL; 24 h,DMEM P/S 2% serum). GED-0301 is an SMAD7 antisense oligonucleotide(GTXGCCCCTTCTCCCXGCAGC, wherein X is 5-methyl-2′-deoxycytidine 5′monophosphate (5-Me-dC) (SEQ ID NO: 6)). Secretion of SEAP induced byTNFα or CD40L in these cells was quantified using QUANTI-Blue™, acolorimetric enzyme assay provided by the manufacturer and specificallydeveloped for this purpose with a medium that changes to a purple-bluecolor in the presence of SEAP, which can be quantified by reading OD at625-655 nm. Data are the average of three independent experiments withquadruplicates per plate using values normalized to the stimulatedresponse (TNFα or CD40L alone) in untreated control cells.

The effects of TGF-β and GED-0301 on NF-κB activation in HEK-Blue cellsby TNFα and CD40L, respectively are shown in FIGS. 2A and 2B. Resultsshow that both TNFα and CD40L produced considerable NF-κB activation (asmeasured by SEAP expression) and these were both suppressed by TGF-β.This suppression was considerably enhanced further by treatment with theSMAD7 antisense oligonucleotide GED-0301 indicating that this treatmentcan enhance and/or restore the effects of TGF-β. In cell culturesystems, TGF-β can either promote or inhibit NF-κB activation dependingon the cell type used (Hong, S. et al. “Smad7 sensitizes tumor necrosisfactor induced apoptosis through the inhibition of antiapoptotic geneexpression by suppressing activation of the nuclear factor-kappaBpathway,” Cancer Res., 2007, 67, 9577-9583 and references therein).TGF-β was a negative regulator of NF-κB in B cells and in humanintestinal lamina propria mononuclear cells (LPMC). The results fortransfected HEK cells are similar to those observed for LPMC, where ithas been shown that TGF-β1 negatively regulates NF-κB activation innormal LPMC (as pretreatment with TGF-β1 suppressed TNF-α-induced NF-κBactivation), and that treatment of IBD LPMC with a specific Smad7antisense resulted in inhibition of NF-κB activation (Monteleone, G. etal. “A failure of transforming growth factor-β1 negative regulationmaintains sustained NF-κB activation in gut inflammation,” J. Biol.Chem., 2004, 279, 3925-3932).

To confirm that GED-0301 can penetrate the assayed cells, HEK Bluesensor cells (50,000/well) were incubated with fluorescein-labeledGED-0301 AS OGN for 48 h in the presence or absence of lipofectamine(LPF, Invitrogen) and then analyzed for cellular uptake. Microscopeimaging confirmed that the HEK Blue sensor cells which were incubatedwith fluorescein-labeled GED-0301 in the presence of lipofectaminedisplayed uptake of the fluorescein-labeled GED-0301 better than cellsnot treated with lipofectamine.

Example 2 Distribution Study of SMAD7 Antisense in Mice

Regular B6 mice (6 wk, males; Jackson Lab.) with multiple low-dosestreptozotocin-induced diabetes (STZ, a β-cell specific toxin; 40 mg/kgi.p. for 5 consecutive days) were administered fluorescein-labeledGED-0301 antisense oligonucleotides (TriLink) by three different routes:oral (p.o.), intraperitoneal (i.p.), and subcutaneous (s.c.) (125μg/mouse=5 mg/kg; p.o. by oral gavage using 500 μL bicarbonate solution,pH 9.5 to protect from gastric degradation). At two differenttime-points (4 h and 24 h), organs were collected and processed from twodifferent animals (one for microscopy, one for flow cytometry). Thefollowing organs were collected: pancreas, liver, kidney, spleen,thymus, intestine, brain, blood, pancreatic lymph nodes, brachial lymphnode, gut-associated lymphoid tissue. For flow cytometry analysis,organs were digested using collagenase D (Roche), passed through a cellstrainer (70 μm; BD Falcon), re-suspended in HBSS, and stained for livecells (LIVE/DEAD® fixable violet dead cell stain kit; Invitrogen) andfor leukocytes (anti-mouse CD45 APC; eBioscience). They were thenanalyzed for fluorescein content m viable cells by using a BD LSR IIFlow Cytometer (BD Biosciences, San Jose, Calif.) and the softwareFlowJo version 7.2.2 (Ashland, Oreg.). FIG. 3A shows an example toillustrate the gating methodology used to analyze the flow cytometryresults. FIG. 3B includes illustrative flow cytometry results obtainedfor the corresponding percent uptakes in viable cells in specificorgans, and they demonstrate significant antisense oligonucleotideuptake (percent FITC+) by the pancreas, pancreatic lymph nodes, andother organs (both in leukocytes, CD45+/FITC+, and in other cells,CD45−/FITC+), especially following s.c. administration. Some organs suchas the brain showed no significant uptake. These results confirm thatGED-0301 can be delivered to specific organs using clinically acceptableadministration routes including subcutaneous administration.

Multichannel high resolution microscopic images (FIG. 4) confirmed thedistribution of GED-0301 into the pancreas of mouse followingsubcutaneous administration of the fluorescently labeled antisenseoligonucleotide in agreement with the flow cytometry results (FIG. 3B).Color legend: green—fluorescent GED0301, grey—DAPI (a marker of cellnucleus), red—insulin (indicating insulin-producing β-cells), andblue—CD45 (indicating CD45 or leukocyte common antigen, LCA, a marker ofwhite blood cells, the elements of the circulating blood system thatcomprise the cells of immunity and inflammation)

Example 3 In Vivo Studies with SMAD7 Antisense

Further in vivo studies can be used to establish the effect of thetreatment in animal models.

Hyperglycemia Reversal Study of SMAD7 Antisense in NOD Mice

Non-obese diabetic (NOD) mice, e.g., NOD mice were used for anintervention-type diabetes prevention trial with treatment started onlyupon onset of hyperglycemia. NOD mice are a commonly used animal modelof type 1 diabetes (Roep, B. O. et al. Satisfaction (not) guaranteed:re-evaluating the use of animal models of type 1 diabetes. Nat. Rev.Immunol., 2004, 4, 989-997), and they have been used to evaluate a largenumber of possible treatments (Shoda, L. K. et al. “A comprehensivereview of interventions in the NOD mouse and implications fortranslation,” Immunity, 2005, 23, 115-126). Eight week old prediabeticfemale animals were procured (Taconic), and starting from week 10(following acclimatization), glycosuria was monitored twice a week. Inanimals that turned positive, blood glucose levels (glycemia) weremonitored three times a week. Animals with elevated glucose levels(nonfasting glycemia>200 mg/dL) on two consecutive days, received onesustained-release insulin pellet implant (LinBit, LinShin Canada, Inc.)to avoid severe hyperglycemia and exhaustion/over-work of the remainingβ-cells; and they were started on treatment. Treatments wereadministered s.c., since this route is clinically relevant and thedistribution studies described above indicated it to be effective intargeting the pancreas, the pancreatic LN, and other organs of possibleinterest. Animals were assigned to one of three treatment arms followinga predefined rotating pattern in the order of their hyperglycemiadevelopment. Treatment (GED-0301=Smad7 AS OGN; 125 μg/mouse) andcorresponding controls (S OGN and saline only) were administered daily.Animals were monitored twice weekly and assessed for their ability tomaintain normoglycemia. Following the disappearance of the effects ofthe implanted insulin pellets (approximately 25 days), animals withthree consecutive glucose readings of >250 mg/dL were considered asdiabetic and sacrificed with representative organs (pancreas, PLN)collected. In animals that did not become diabetic, daily treatment wasstopped after 10 weeks, and they were monitored for an additional 12weeks (up to a total of 150 days) to establish whether there is alasting effect. At the end, all animals were sacrificed andrepresentative organs (pancreas, PLN, and others) collected.

Results from the study are shown in FIG. 5 with blood glucose levels inFIG. 5A and a corresponding Kaplan-Meier survival curve showing thepercent of animals remaining diabetes-free as a function of time in FIG.5B. Treatment was initiated after the onset of hyperglycemia when β-celldamage had already occurred. Accordingly, severe hyperglycemia candevelop very quickly. All animals treated with saline or senseoligonucleotide control developed diabetes (n=7 and 6, respectively), asindicated by their hyperglycemia (blood glucose>250 mg/dL) following theexhaustion of the LinBit implant with sustained release insulin by day20-25. For the animals treated with the active GED-0301 antisenseoligonucleotide, 6 out of the 11 treated did not develop hyperglycemiaand they remained diabetes-free up to the end of the study (day 150),long after the treatment had been stopped (day 70).

To verify the glucose response of non-diabetic animals at the end of thestudy (day 150), an oral glucose tolerance test (OGTT) was performed inseveral GED-0301-treated as well as control mice. A glucose dose of 150mg was administered by oral gavage to mice fasted for 16 h and bloodsamples were collected at predefined time intervals for up to 150minutes. Results shown in FIG. 6 confirm that the glucose response, andhence insulin-secreting ability, was comparable to that of healthyanimals. Blood glucose levels returned to normal within 150 minutes ofthe oral challenge in all animals.

High-resolution confocal microscopic imaging of pancreas sections fromGED-0301-treated NOD mice that did not become diabetic by the end of thestudy (day 150) was performed to confirm the presence ofinsulin-producing β-cells. FIG. 7 shows a series of multichannel Z-stackimages of representative islet-containing sections of pancreas fromGED-0301-treated mice and corresponding controls. Different colors(columns) are as follows: blue is DAPI (a marker of cell nucleus), redis insulin (indicating insulin-producing β-cells), and green is glucagon(indicating glucagon-producing α-cells). The scale bar indicates 100 μm.Whereas normal, non-diabetic control animals had normal isletscontaining both insulin- and glucagon-producing cells (top row), NODmice that became diabetic during the study lost the insulin-producingβ-cells from their islets (bottom row; from a senseoligonucleotide-treated mouse that did not revert to normoglycemia).GED-0301 antisense (AS) treated animals that did not become diabeticfollowing treatment initiated at the onset of hyperglycemia had severalislets that lost their insulin-producing cells (second row from topshowing the presence of glucagon, but not insulin), but they also hadislets with conserved insulin-producing cells (third row from top).Hence, GED-0301 treatment initiated at the onset of hyperglycemia seemedto be able to counter the effects of autoimmune attack in NOD mice andpreserve insulin-producing β-cells in sufficient numbers to maintainnormal blood glucose response and avoid the onset of hyperglycemia.

Study of SMAD7 Antisense in Islet Transplantation

Restoration of TGF-β signaling in the local microenvironment has shownpromise in experimental allogeneic islet transplantation models,including in the presence of an underlying autoimmune disease. Treatmentwith GED-0301 can be done to establish the effect of an antisensetargeting SMAD7 on the survival and/or tolerance to allogeneic isletgrafts. Specifically, the effect of GED-0301 in preventing rejectionand/or prolonging survival of islets in a model of allogeneictransplantation in rodents rendered diabetic by the administration of abeta cell-specific toxin (STZ) can be determined. Primary endpoints caninclude islet allograft survival, activation markers on T lymphocytes,dendritic cells, frequency and suppressive function of Treg and othersuppressor cells as a correlation with Smad7 expression/modulation inthe transplantation site.

Study of SMAD7 Antisense and Low-Grade Inflammation in Type 2 Diabetes

As low-grade systemic and local inflammation characterizes prediabetesand type 2 diabetes, the potential role of SMAD7 in the development ofthe low-grade inflammation within the pancreatic islet can bedetermined. Furthermore, the role of SMAD7 in islet vascularcomplications in diabetes can be studied.

Study of SMAD7 Antisense in Diabetic Nephropathy

In human diabetic nephropathy and other proteinuric glomerular diseases,Smad7 is strongly upregulated. This upregulation occurs primarily inpodocytes, the glomerular cells responsible for the integrity of theglomerular filtration barrier and for the prevention of albuminuria, anindependent predictor of cardiovascular outcome in the generalpopulation. Targeting of TGF-beta has been the topic of an intensetranslational effort (trial with pirfenidone). In these efforts, resultshave been unexpectedly negative for the cure of diabetic nephropathy andother chronic kidney diseases. Restoring a proper TGF-β signaling bySmad7 inhibition may be a promising and novel approach to treat theseconditions. Established experimental models of diabetic nephropathydeveloped by the NIH Consortium on Diabetic Complications can beutilized to generate preliminary in vivo data on soft and hard renaloutcome (albuminuria, glomerular filtration rate, glomerulosclerosis).Subsequent clinical studies can be developed in the Diabetic Nephropathyclinic at the Diabetes Research Institute.

Study of SMAD7 Antisense on Glucose Stimulated Insulin Secretion

Mice with pancreatic β-cells with Smad7 overexpression are characterizedby impairment of insulin release and increased fasting glucose.Therefore, Smad7 inhibition could result in the improvement ofpancreatic β-cell function. Glucose-stimulated insulin-releaseexperiments in human islets genetically manipulated to express differentlevel of SMAD7 can be performed. In further studies, human patients canbe studied to determine the role of Smad7 inhibition in glucoseintolerance in pre-diabetic subjects.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles cited herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention can be embodied in other specific forms with departingfrom the essential characteristics thereof. The foregoing embodimentstherefore are to be considered illustrative rather than limiting on theinvention described herein. The scope of the invention is indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

The invention claimed is:
 1. A method of treating diabetes by preventingpancreatic islet β cell destruction, the method comprising administeringto a patient in need thereof an effective amount of a SMAD7 antisenseoligonucleotide comprising a sequence selected from the group consistingof the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, and SEQ ID NO: 12, wherein the diabetes is selected fromthe group consisting of Type 1 diabetes, Type 2 diabetes, andgestational diabetes.
 2. The method of claim 1, wherein the SMAD7antisense oligonucleotide comprises the nucleotide sequence of SEQ IDNO: 6 or SEQ ID NO:
 10. 3. The method of claim 1, wherein the SMAD7antisense oligonucleotide is administered parenterally.
 4. The method ofclaim 3, wherein the SMAD7 antisense oligonucleotide is administeredsubcutaneously.
 5. The method of claim 1, wherein the SMAD7 antisenseoligonucleotide comprises the nucleotide sequence of SEQ ID NO:
 10. 6.The method of claim 5, wherein all internucleoside bonds of the SMAD7antisense oligonucleotide comprising the nucleotide sequence of SEQ IDNO: 10 are phosphorothioate bonds.
 7. The method of claim 1, wherein twoor more of the internucleoside bonds of the SMAD7 antisenseoligonucleotide comprising SEQ ID NO: 10 are phosphorothioate bonds. 8.A method of treating diabetes by preventing pancreatic islet β celldestruction, the method comprising administering to a patient in needthereof a pharmaceutical composition comprising: an effective amount ofa SMAD7 antisense oligonucleotide comprising a sequence selected fromthe group consisting of the nucleotide sequences of SEQ ID NO: 3, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12; and apharmaceutically acceptable carrier, wherein the diabetes is selectedfrom the group consisting of Type 1 diabetes, Type 2 diabetes, andgestational diabetes.
 9. The method of claim 8, wherein the SMAD7antisense oligonucleotide comprises the nucleotide sequence of SEQ IDNO: 6 or SEQ ID NO:
 10. 10. The method of claim 8, wherein the SMAD7antisense oligonucleotide comprises the nucleotide sequence of SEQ IDNO:
 10. 11. The method of claim 10, wherein all internucleoside bonds ofthe SMAD7 antisense oligonucleotide comprising the nucleotide sequenceof SEQ ID NO: 10 are phosphorothioate bonds.
 12. The method of claim 8,wherein the pharmaceutical composition is administered parenterally. 13.The method of claim 12, wherein the pharmaceutical composition isadministered subcutaneously.
 14. The method of claim 8, wherein two ormore of the internucleoside bonds of the SMAD7 antisense oligonucleotidecomprising SEQ ID NO: 10 are phosphorothioate bonds.
 15. A method oftreating hyperglycemia by preventing pancreatic islet β celldestruction, the method comprising administering to a patient in needthereof an effective amount of a SMAD7 antisense oligonucleotidecomprising a sequence selected from the group consisting of thenucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 10, and SEQ ID NO:
 12. 16. The method of claim 15, wherein the SMAD7antisense oligonucleotide comprises the nucleotide sequence of SEQ IDNO:
 10. 17. The method of claim 16, wherein all internucleoside bonds ofthe SMAD7 antisense oligonucleotide comprising the nucleotide sequenceof SEQ ID NO: 10 are phosphorothioate bonds.
 18. The method of claim 15,wherein the SMAD7 antisense oligonucleotide is administeredparenterally.
 19. The method of claim 18, wherein the SMAD7 antisenseoligonucleotide is administered subcutaneously.
 20. The method of claim15, wherein two or more of the internucleoside bonds of the SMAD7antisense oligonucleotide comprising SEQ ID NO: 10 are phosphorothioatebonds.
 21. A method of treating hyperglycemia by preventing pancreaticislet β cell destruction, the method comprising administering to apatient in need thereof a pharmaceutical composition comprising: aneffective amount of a SMAD7 antisense oligonucleotide comprising asequence selected from the group consisting of the nucleotide sequencesof SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ IDNO: 12; and a pharmaceutically acceptable carrier.
 22. The method ofclaim 21, wherein the SMAD7 antisense oligonucleotide comprises thenucleotide sequence of SEQ ID NO:
 10. 23. The method of claim 22,wherein all internucleoside bonds of the SMAD7 antisense oligonucleotidecomprising the nucleotide sequence of SEQ ID NO: 10 are phosphorothioatebonds.
 24. The method of claim 21, wherein the pharmaceuticalcomposition is administered parenterally.
 25. The method of claim 24,wherein the pharmaceutical composition is administered subcutaneously.26. The method of claims 21, wherein two or more of the internucleosidebonds of the SMAD7 antisense oligonucleotide comprising SEQ ID NO: 10are phosphorothioate bonds.